WO2010030823A1

WO2010030823A1 - Mixed microorganism communities for the production of biomass

Info

Publication number: WO2010030823A1
Application number: PCT/US2009/056570
Authority: WO
Inventors: Anthony Michaels; Dave Caron
Original assignee: Phycosystems Inc.
Priority date: 2008-09-10
Filing date: 2009-09-10
Publication date: 2010-03-18

Abstract

A method for producing large amounts of organic matter using a complex mixture of multiple strains of microalgae, protists, bacteria and other organisms to create a consistent, rapidly- growing managed ecological community under multiple or varying environmental conditions that is resistant, by design, to failures through invasion or disease. The method is optimized for the production of feedstock for renewable fuels through gasification and is also adaptable to production of biomass for other biomass-based fuels, animal feeds, fish feeds and other needs for large amounts of quality organic matter.

Description

MIXED MICROORGANISM COMMUNITIES FOR THE PRODUCTION OF BIOMASS

5 BACKGROUND

[0001] Microalgae are well known as some of the fastest growing autotrophic organisms and have been promoted as solutions for producing organic foods and renewable fuels. Most existing approaches emphasize selection of one or, at most, a few optimum strains based on growth rates and biochemical composition (e.g. oil content) and their incubation in defined,

10 presumably optimum conditions. These existing approaches must hold the organisms in acceptable growing conditions and must guard against biological failures like invasion of another alga, contaminants and disease. Historically, these methods have been both expensive to follow and difficult to operate consistently, contributing to a high cost of the final algal product. The invention described herein solves these problems by providing an

15 inexpensive bioreactor system and method which provides a very consistent operation with high yield.

SUMMARY OF THE INVENTION

[0002] The new approach embodied in this invention exploits the ability of carefully

_7n selected communities of organisms (complex mixtures of organisms) to internally self-select the strains that will grow best under each condition, maximizing net community production under varying conditions. It is resistant to invasion, because the success of a newly introduced strain becomes just another part of the success community and gets incorporated into the mix. Diseases or grazers that target one, successful algae or a subset of the

, - community merely open the community to success by another of the algal species in the community. Bacteria convert leaked dissolved organic matter into biomass and also produce some of the required micronutrients that the algae cannot make themselves. The result is a robust, resilient community of opportunistic algae and related enhancing organisms that yield a consistently rapid rate of biomass creation. This method provides an approach tailored for

_™ each specific bio-reactor and growing environment to allow for an optimal solution in many different engineered or managed environments.

BRIEF DESCRIPTION OF DRAWINGS

[0003] The foregoing invention will be better understood by reference to the drawings wherein:

35 [0004] Figure 1. A schematic diagram of multiple strains of algae for providing consistently high production rates of algae under any combination of conditions. 1 [0005] Figure 2. A schematic diagram of a bioreactor system according to the present invention used to hold the complex ecological community and to extract the biomass product produced by the system and method of the present invention.

₅ DETAILED DESCRIPTION OF THE INVENTION

[0006] The invention is a unique biological system comprised of a complex biological community that, by design, always has algae that grow rapidly and fill any ecological gaps that might compromise its growth. As conditions change, this designed, complex community substitutes new, better strains from a pool of rarer algae that are always present in the n background. Invasive algae become incorporated into the system as an improvement rather than a contaminant. Disease organisms that strike a single strain (like a virus) open up a niche and are replaced by a similar alga that was being out competed by the target of the disease. The invention also provides a method for customizing the system for any specific bio-reactor and environmental setting.

, j. [0007] A combination of .one or more algae such as Prymnesium, Dunaliella,

Micromonas, Rhodomonas, Phaeocystis and Nitzschia, protists such as Uronema and Euplotes and bacteria such as Pseudomonasaτe added to a bioreactor which can be one of any of a variety of existing conventional designs. All algae are grown in a container that holds the water and nutrients with access to light and creates a unique set of environmental

^₀ conditions for the algae in that water. These containers are called bioreactors and the present invention can function in any form of bioreactor in any environment through the customization process described herein. The combination of algae, protists and bacteria includes organisms that are specifically selected to cover the range of conditions that are anticipated in the bioreactor and in alternate embodiments will also include some redundancy _{j c} (organisms with similar strengths) and some generalists.

[0008] In one embodiment, the community includes at least one species of algae (e.g. Prymnesium) that will grow well at the highest anticipated temperature, at least one species (e.g. Nitzschia) that grows well at the lowest anticipated temperature, at least one species (e.g. Dunaliella) that grows well at the initial nutrient concentration, at least one species (e.g.

~~ Rhodomonas or Micromonas) that grows well at relatively low nutrient concentrations and at least one species (e.g. Phaeocystis) that is known as a weedy, invasive species. The mixture also includes at least one bacterium (e.g. Pseiidomonas) that efficiently converts dissolved organic matter to biomass or one bacterium (e.g. a different Pseudomonas species) that makes a critical organic nutrient such as a B vitamin. The system includes one or more

, , heterotrophic protists to convert bacteria and ultra-small algae into larger biomass, one of which consumes planktonic species and the other a specialist which consumes surface dwelling species (e.g. Uronema and Euplotes). In another embodiment a mixotroph such as Prymnesium is added to achieve these objectives in a single strain. In an alternative

-9- embodiment, the mixture includes a blend of 10-15 species that are known to form large, dense algae blooms in natural settings that emulate the environmental ranges as seen in the bioreactor. In another alternative embodiment, the mixture includes algae that produce allelopathic chemicals that slow the growth rates of its competitors. Some embodiments may include all of these species. Different species fill each role or other unique roles are presented by the basic condition in the bioreactor.

[0009] Figure 1 illustrates four algae that each have a specific, measurable growth response to light and nutrients. [0010] Alga A grows well on high light and high nutrients and Alga D grows well on low light and low nutrients. Alga B grows well in high nutrients, but is slowed when light is high (it is adapted to low light). Alga C grows well in high light, but is adapted to low nutrients and grows slow when nutrients are high. Thus, there are four "specialists" for each combination of circumstances. When a bioreactor operation is initiated, normally there are high nutrients present and, because there are few algae, the light is high; thus Alga A will grow fast and dominate the community. As the algae grow and the light levels drop from shading, Alga B will begin to take over. As nutrients are used up, Alga D starts to flourish. At the time of harvest or if the algae settle, there will be periods of high light (cleared water) and low nutrients where Alga C thrives. The complex ecosystem design of the present invention provides a new organism for each set of circumstances in the life of the community in the bioreactor so as to always have a fast growing total community. In other embodiments of this invention, the community includes strains that cover other environmental conditions that the bioreactor might encounter that would slow growth, including temperature, contaminants, trace metal presence, stresses of a pumping system or transport in tubes and bags, the presence of other organisms or allelopathic compounds and other conditions. These will be determined experimentally or by specific choice.

[0011] In a closed and isolated setting, these species are drawn from publicly available culture collections. However in a more open setting or one that is ecologically sensitive, the system relies on selecting species from the natural environment proximate to the area of use. The local strains are then analyzed and selected for the characteristics that allow them to fill the specific ecological role desired.

[0012] As indicated previously, all algae are grown in a container called a "bioreactor." In one embodiment a bioreactor is a simple open topped water containing vessel that is of a selected size (from a few liters to many cubic kilometers) with a transparent or open top that allows most or all of the ambient sunlight to reach the surface of the water. Water is provided at a sufficient depth for the desired net biological production rate. Also included in the bioreactor is a predetermined supply of carbon dioxide and nutrients to ensure maximum growth for the intervals between sampling or nutrient supply. Preferably the bioreactor includes automated systems for the addition of carbon and nutrients to ensure an adequate supply of each. The bioreactor circulates the water both vertically and horizontally to keep organisms suspended, providing reasonable access to light and keeping the nutrients well mixed into the surface layer. The circulation mechanism comprises low-disturbance devices like paddles, or in the alternative pumps, to minimize disturbance to the organisms or to set a specific level of disturbance depending on the biological community. A number of existing bioreactors are well known and are suitable for use in the present invention. For example, raceways are a type of open pond that uses an oval shape and paddlewheel system to move the water along a predetermined path. Such raceways are already in commercial use for culturing of Spirulina in California, Hawaii, Asia and elsewhere. Open ponds are the simplest form of bioreactor. Other more complex bioreactors can be utilized which are fully enclosed systems with tubes or bags holding the water and nutrients and managing light access.

[0013] As shown in Fig. 2, the elements of the total system may include an algae bioreactor which is operatively connected to a dewatering system from which the dried algae biomass end product is extracted.

[0014] The actual physical location of the bioreactor must have known environmental conditions as these conditions affect the water in the incubator, both when it has little biomass and when it is near its peak biomass and relevant points in between. The environmental conditions that must be considered include the following: the temperature patterns, evaporation patterns, the amount and variability of the light environment, the nature of the nutrient, trace metal and organic contaminant content of the source feed water and the composition of any carbon and nutrient feed-stocks.

[0015] At the initiation of operation of the bioreactor and practice of the method according to the present invention, data for all of the foregoing conditions and other relevant parameters are considered including hourly data for an entire year coupled with an understanding of how these data might vary with normal climate fluctuations. The data can be compiled from any combination of measured, estimated and modeled results. To these is added a database of the basic physiology and growth-rate characteristics of the microalgae, protists and bacteria used in the compositions. These data are derived from a combination of direct measurements, literature and first principles. Examples of suitable microalgae are Dunaliella, Phaeocystis, Prymnesium, Nitzschia, Emialiania, Spyrogyra and Rhodomonas. Examples of suitable heterotrophic protists are Uronema, Euplotes and Paraphysomonas. Examples of suitable bacteria are Microcystis, Oscillatoria, Anabena, Spirulina and Pseudomonas. [0016] The bioreactors are filled with either freshwater, brackish water or seawater of known composition. Major and micro nutrients and carbon are added at the beginning and at preselected intervals thereafter. Examples of major nutrients are nitrate, ammonia, phosphate, silicate and carbon dioxide. Examples of micro-nutrients are iron, manganese, zinc, copper, cobalt, vitamins, etc. Carbon may be added in the form of carbon dioxide, bicarbonate, carbonate and/or carbonic acid.

[0017] The bioreactor is provided with instrumentation to monitor the range of environmental conditions referred to above. [0018] In order to refine the operating conditions most appropriate to a specific bioreactor setting, an iterative testing procedure is used. The same initial mix of species is added to multiple bioreactors for replication and verification of each combination of organisms. The combination of bioreactors is referred to as a bioreactor test set.

[0019] The mixed community of organisms is incubated in each bioreactor test set and its growth rate and species composition are monitored. Additional species are added to supplement conditions where the initial blend does not have one or more rapidly growing species. Species that disappear are noted. Particularly successful combinations are noted and new bioreactor test sets are started with those combinations. Some test sets are challenged by introducing new strains of opportunistic or invasive algae. This pattern is repeated iteratively to arrive at combinations of species that give rapid growth rates under varying conditions and are resistant to invasion by other opportunistic species.

[0020] Optimized versions of this complete algae community are then installed into production bioreactors which in turn have been preprogrammed to yield a complex ecological community that has at least one ecological winner — a strain that grows better than the others. The remaining strains exist at low concentrations until some change in environment or circumstances allows them to be the winner and take over the dominant biomass production role.

[0021] The bioreactors can either be am in batch mode or in continuous mode with regular addition of new nutrients and continuous removal of biomass or a combination of the two.

[0022] As the algae reach high levels of biomass, they are harvested in the dewatering system of Fig. 2 by one or more established techniques including coagulation, settling, centrifugation and filtration.

[0023] In another embodiment, genetic modifications are made to some of the organisms to enhance a specific characteristic of their growth, such as using quorum sensing to cause the release of flocculants at appropriate concentrations to augment the concentration of the algae, the turning off or enhancement of specific physiologies like lipid or starch production or other characteristics of key elements of the community.

[0024] In other embodiments of the invention, algae like Emiliania are added to the community in the bioreactor to make a calcium carbonate skeleton which can then be harvested separately as a carbon sequestration product.

Claims

WHAT IS CLAIMED IS:

1. An artificial community of micro-organisms comprising: a) two or more algae species, each of which is selected to thrive in one or more environmental conditions or one or more combinations of environmental conditions in a bioreactor; b) protists selected for their ability to promote biomass increase; c) bacteria selected for their ability to promote conversion of dissolved organic matter to biomass; combined in the bioreactor, wherein the community grows and maintains a high amount of biomass and the total biomass is resistant to failures through disease, invasion or other biological interruptions.

2. A community according to claim 1, wherein the algae include at least one species of algae that is adapted to thrive in each of the various environmental conditions or combinations of various environmental conditions.

3. A community according to claim 1, wherein the algae include: a) at least one species that grows well at the highest anticipated temperature in the bioreactor; b) at least one species that grows well at the lowest anticipated temperature in the bioreactor; c) at least one species that grows well at relatively high nutrient concentrations in the bioreactor; d) at least one species that grows well at relatively low nutrient concentrations in the bioreactor; e) at least one species that grows well at high light levels; f) at least one species that grows well at low light levels; and g) at least one species that is a weedy, invasive species.

4. A community according to claim 1 , wherein the protists are selected to consume organisms that are harmful to the community.

5. A community according to claim 1, wherein the bacteria are selected to consume dissolved organic matter and produce biomass or generate a nutrient.

6. A community according to claim 1, the community further comprising one or more algae that produce allelopathic chemicals that slow the growth rate of competing organisms.

7. A bioreactor system comprising: a container wherein water, nutrients and a source of light are present; to which is added two or more microalgae and at least one organism which produces allelopathic chemicals selected from the group consisting of microalgae, heterotrophic protists, and bacteria; wherein the microalgae and the at least one organism are selected in light of conditions encountered internally and externally of the bioreactor to prevent failure of the biomass and maximize growth in varying conditions.

8. The system of claim 7, wherein the microalgae in the system include a species that grows well at the highest anticipated temperature in the bioreactor, a species that grows well at the lowest anticipated temperature in the bioreactor, a species that grows well at high nutrient concentrations in the bioreactor, a species that grows well at relatively low nutrient concentrations in the bioreactor, a species that grows well at high light levels, a species that grows well at low light levels, and a species that is a weedy, invasive species.

9. The system of claim 7, further comprising at least one heterotrophic protist species, wherein the at least one heterotrophic protist species consumes organisms that are harmful to the community.

10. The system of claim 7, further comprising at least one bacterium species, wherein the bacterium species consumes dissolved organic matter and produces biomass or generate a nutrient.

11. The system of claim 7, further comprising at least one bacterium species and at least one heterotrophic protist species, wherein the at least one bacterium species consumes dissolved organic matter and produces biomass or generates a nutrient and wherein the at least one protist species consumes organisms that are harmful to the community.

12. A method of producing large amounts of biomass comprising: a) creating a bioreactor; b) measuring environmental conditions affecting the bioreactor; c) creating a community comprising two or more algae and at least one organism selected from the group consisting of protists, bacteria, an organism which produces allelopathic chemicals, and combinations thereof, wherein the two or more algae and the at least one organism are selected to maximize net biomass production under the measured environmental conditions present at the bioreactor, and to be resistant to invasion, disease, or other biological interruptions

13. The method of claim 12, further comprising: d) introducing water, nutrients, and carbon into the bioreactor; e) monitoring the water, nutrient, and carbon levels in the bioreactor; and 0 adding additional water, nutrients, and carbon to the bioreactor in response to changes in the water, nutrient, and carbon levels in the bioreactor.

14. The method of claim 12, further comprising monitoring the environmental conditions at the location of the bioreactor and adding at least one additional organism selected from the group consisting of algae, protists, bacteria, an organism which produces allelopathic chemicals, and combinations thereof in response to changes in the environmental conditions at the location of the bioreactor, wherein the additional organism is selected to cause the biomass to thrive in the changed environmental conditions or combination of environmental conditions.

15. The method of claim 12, further comprising monitoring the growth of the species of algae in the biomass in the bioreactor and adding at least one additional organism selected from the group consisting of algae, protists, bacteria, an organism which produces allelopathic chemicals, and combinations thereof so that at least one species of algae in the biomass is rapidly growing.

16. The method of claim 12, further comprising removing at least a portion of the biomass produced in the bioreactor.

17. The method of claim 12, further comprising extracting at least a portion of the biomass produced in the bioreactor using a dewatering system and returning the water removed from the extracted portion of the biomass to the bioreactor.